N cad

Western blotting analysis was used to perform the protein expression of relative genes. Briefly, the pretreated EC109 were lysed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitors cocktail (Jas enzyme, China). The supernatant was used to detect the concentration of protein by BCA protein assay Kit (Beyotime, China). Glut1, HK2, LDH, TLR4, p-Akt, HIF-1α, Wnt, β-catenin, N-cad, E-cad, Vamentin (Proteintech, China) primary antibodies were diluted 1000 times and β-actin diluted 5000 times were used in western blotting. The goat anti-rabbit or anti-mouse IgG secondary antibody was diluted by 1:5000. Subsequently, Omni-ECL™pico pight chemiluminescence kit (Jas enzyme, China) was used to analyze the protein brand under enhanced chemiluminescence system (Tanon, China). Band intensities from three biological experiments were quantified by densitometry using ImageJ software.

IHC was performed as described previously [19 (link), 20 (link)]. Briefly, tissue sections were deparaffinized and dehydrated, incubated with H₂O₂, subjected to high-pressure repair, and blocked with BSA. anti-Numb (Abcam, Cambridge, UK, dilution: 1:400), Snail1 (Proteintech, Chicago, IL, dilution: 1:200), Snail2 (Proteintech, dilution: 1:200), E-cad (Abcam, 1:1000), N-cad (Proteintech, dilution: 1:200), Vimentin (Proteintech, dilution: 1:500), and Ki67 (Proteintech, dilution: 1:500) overnight. Slices were covered with the secondary antibody, detected with DAB, co-stained with hematoxylin, and evaluated by two professional pathologists for the staining scores.